text: NS5A inhibitors have been developed to target the NS5A protein. These inhibitors have achieved a significant reduction in HCV RNA blood levels and can therefore be considered as potent antivirals. Their mechanism of action is thought to be diverse but the exact mechanism is not fully understood. Most studies assume that NS5A inhibitors act on two essential stages of the HCV life cycle; the replication of the genomic RNA, and virion assembly. Other studies propose an alteration of host cell factors as a possible third mechanism.
  The structure of NS5A inhibitors is characterized by dimeric symmetry. This suggests that NS5A inhibitors act on dimers of NS5A. A number of modeling studies have shown that daclatasvir, which is an NS5A inhibitor, only binds to the "back-to-back" NS5A dimer and that the binding has to be symmetrical. Other modeling studies have shown that binding to other conformations of NS5A might be possible, as well as asymmetrical binding. Research has shown that daclatasvir's target is most likely domain I of NS5A. Even though the mechanism is not completely understood, it has been demonstrated that the inhibitors downregulate NS5A hyperphosphorylation, leading to the suppression of HCV replication and its processing of polyproteins, as well as resulting in an unusual protein location. Hitherto, this inhibition was thought to require only NS5A domain I, but not domains II and III. However, recent studies have shown that both domains I and II are relevant to this disruption of RNA replication.
  NS5A inhibitors appear to furthermore disrupt the formation of new replicase complexes resulting in a gradual slowing of viral RNA synthesis. Effect on previously formed complexes has yet to be demonstrated.
  Available evidence suggests that NS5A inhibitors modify the location of NS5A inside the cell. This may cause abnormal assembly leading to malformed viruses. Some studies have revealed that inhibition of the viral assembly has a more important role in RNA reduction than viral replication reduction.
  Studies have shown that NS5A inhibitors block the formation of the membranous web, which protects the viral genome and features the main sites for viral replication and assembly. This mechanism is thought to be independent of RNA replication, but seems to be affected by NS5A inhibitors blocking the formation of the PI4KIIIα-NS5A complex, essential to the synthesis of the PI4P, resulting in decreased integrity of the membranous web and therefore reduced HCV RNA replication.